Nearly 50 million Americans smoke tobacco and smoking is the leading cause of premature death and disability in the US. Although the transition from tobacco use to nicotine dependence is associated with enduring changes in multiple motivational mechanisms, most neurobiological research has focused on reward- related systems. Very little attention has been devoted to understanding the brain circuits involved in withdrawal-related negative affect in humans. This is unfortunate?heightened anxiety is a hallmark of nicotine deprivation in both rodents and humans and there is compelling evidence that this evolutionarily-conserved feature of withdrawal powerfully motivates nicotine dependence and relapse. Mechanistic work in rodents and imaging work by our group suggests the hypothesis that withdrawal-potentiated anxiety reflects alterations in a neural circuit encompassing the bed nucleus of the stria terminalis (BNST), anterior insula (AI), and mid- cingulate cortex (MCC). But the relevance of this circuitry to withdrawal in humans is unexplored and remains unknown. Leveraging our team's unique multi-disciplinary expertise, the goal of this proposal is to use an innovative combination of advanced fMRI analytic techniques and mobile phone-based ecological momentary assessment (EMA) to understand, for the first time, the relevance of anxiety-related brain circuits to chronic tobacco use and acute nicotine withdrawal in abstinent smokers. fMRI will be used to assay anxiety-related activation and functional connectivity in well matched groups of 24-hour abstinent and non-abstinent tobacco smokers (n=36/group; half female). EMA will be used to assay stressor exposure, negative affect, smoking urge/craving, alcohol/substance use, and smoking for 14 days (5x/day), including the 24-hour period of withdrawal immediately prior to the fMRI session. These data would enable us to systematically understand the relevance of anxiety-related brain circuits to: (1) acute nicotine withdrawal and (2) clinically-relevant features of mood and behavior in the real world. These objectives are closely aligned with the NIDA Strategic Plan. Addressing the first objective would afford an unprecedented opportunity to examine the translational relevance of prominent neurobiological models of addiction, guide the development of bidirectional translational models of nicotine dependence, identify new biological targets at the circuit level, and inform the develop of novel cessation aids. Addressing the second objective would refine our understanding of etiology, provide new targets for behavioral therapy, and inform the development of mobile phone-based interventions. Understanding the role of anxiety-related brain circuits in withdrawal is critical for efforts aimed at reducing the tremendous suffering and economic burden caused by tobacco dependence.